Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation. Cancer therapies often target specific proteins within pathways of biochemical networks. Precision medicine depends on the accurate diagnosis of disease using a variety of clinical assays to guide the selection of appropriate therapies 1. Diagnostic assays must be performed on high quality tissue where the biochemical networks are preserved and representative of the biological state of the highly dynamic signaling network. One example of a precision medicine test is immunohistochemistry (IHC), such as the test for BRAF V600E mutation using the VE1 clone IHC used in colon, thyroid, and melanoma for prognostic value to guide treatment decisions 2,3. BRAF V300E assays, like all IHC assays, are susceptible to preanalytical errors such as inadequate fixation or tissue processing, yet fixation and processing steps are often de-emphasized or taken for granted as an established part of the hospital workflow 1. Formalin fixation has been used for more than a century to preserve tissue for histopathology, our understanding of the biochemistry of formalin fixation is incomplete. There is evidence that cold formalin fixation improves the preservation of biochemical markers especially within signaling networks such as phosphoproteins 4,5 , and that cold formalin fixation has been shown to aid in the preservation of nucleic acids 6. The rapid two-temperature (2 + 2) protocol was developed to take advantage of the biochemistry of formaldehyde in solution, where methylene glycol exists in equilibrium with formaldehyde in a temperature dependent manner 4. At lower temperatures (4 °C), nonreactive methylene glycol is present in great excess o...